Functional Role of Pseudouridine on Zika Virus Gene Expression

寨卡病毒(ZIKV)是一种重新出现的蚊媒黄病毒，在2015-2016年美洲爆发期间，它与严重和毁灭性的发育异常有关，包括由受感染妇女出生的婴儿的小头畸形。ZIKV的单链正义RNA基因组指导病毒蛋白质的翻译，作为基因组复制的模板，并被包装成新的病毒颗粒。这些功能在一定程度上受到RNA非翻译区内特定RNA结构的调节，并通过与病毒和细胞蛋白的相互作用来调节。最近，RNA修饰已被证明显著影响病毒的感染周期。虽然已知有170多种不同的RNA修饰天然RNA，但只有N6-甲基腺苷被报道影响ZIKV基因的表达。其他RNA修饰对ZIKV RNA的作用目前尚不清楚。利用生物素化的反义寡核苷酸从细胞和病毒粒子中亲和分离病毒RNA，并用质谱仪鉴定RNA修饰，我们发现ZIKV RNA上存在30多种不同的RNA修饰。在这一应用中，我们主要关注伪尿苷(Y)，这是一种含量丰富但研究较少的RNA修饰。Y在细胞RNA上的掺入影响RNA的折叠、稳定性和翻译，以及RNA-RNA和RNA-蛋白质的相互作用。我们认为ZIKV RNA基因组上的Y促进病毒的翻译和复制。在这项应用中，我们建议使用下一代测序方法结合伪尿嘧啶的化学修饰来识别ZIKV基因组上的特定Y位点(目标1)。接下来，我们将进行RNAi耗尽和细胞伪尿苷合成酶蛋白的过表达，以确定负责在ZIKV RNA上安装伪尿嘧啶的宿主酶(目标2)。最后，我们将使用假尿苷合成酶的突变和调制来阐明假尿苷是否调节ZIKV的翻译和/或复制(目标3)。此外，这些研究将在哺乳动物和蚊子细胞中病毒感染的背景下进行，这两个宿主在生物学上与病毒生命周期相关，这可能会揭示RNA修饰在病毒传播和宿主适应中的作用。我们希望这项研究将促进我们对RNA修饰的了解，因为RNA修饰已被证明是病毒基因表达的新的和重要的调节因素，并提高了我们对ZIKV分子生物学的理解。因此，这项研究将提供有关这种病毒的关键信息，这种病毒对人类健康有重大影响，目前缺乏治疗选择。

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus, that during the 2015-2016 outbreak in the Americas was associated with severe and devastating developmental abnormalities, including microcephaly, in babies born from infected women. The single-stranded positive-sense RNA genome of ZIKV directs translation of viral proteins, functions as a template for replication of the genome and is packaged into new virus particles. These functions are in part regulated by specific RNA structures within the untranslated regions of the RNA, and via interactions with viral and cellular proteins. More recently RNA modifications have been shown to significantly affect the virus infectious cycle. Although more than 170 different RNA modifications are known to decorate natural RNAs, only N6-methyladenosine has been reported to affect ZIKV gene expression. The role of other RNA modifications on ZIKV RNA is presently unknown. Using biotinylated antisense oligonucleotides to affinity isolate virus RNA from cells and virions, and mass spectrometry to identify RNA modifications, we discovered that more than 30 different RNA modifications are present on ZIKV RNA. In this application we focus on pseudouridine (Y), an abundant but understudied RNA modification. The incorporation of Y on cellular RNAs affects the folding, stability, and translation of RNA, and RNA-RNA and RNA-protein interactions. We propose that Y on the ZIKV RNA genome promotes virus translation and replication. In this application we propose using a next generation sequencing approach combined with chemical modification of pseudouridine to identify specific Y sites on ZIKV genome (Aim 1). Next, we will undertake RNAi depletion and overexpression of cellular pseudouridine synthase proteins to identify the host enzymes responsible for installing pseudouridine on ZIKV RNA (Aim 2). Last, we will use mutagenesis and modulation of the pseudouridine synthase enzymes to elucidate if pseudouridine regulates ZIKV translation and/or replication (Aim 3). Moreover, these studies will be undertaken in the context of virus infection in mammalian and mosquito cells, two hosts biologically relevant to the virus life cycle, which could inform the role of RNA modifications in virus transmission and host adaptation. We expect that this research will advance our knowledge of RNA modifications which have been shown to be novel and important regulators of viral gene expression and improve our understanding of the molecular biology of ZIKV. This research will therefore provide critical information on a virus with significant impacts on human health and that currently lacks therapeutic options.